Cthrc1 deficiency aggravates wound healing and promotes cardiac rupture after myocardial infarction via non-canonical WNT5A signaling pathway

Cardiac fibroblasts are crucial for scar formation and cardiac repair after myocardial infarction (MI). Collagen triple helix repeat containing 1 (CTHRC1), an extracellular matrix protein, is involved in the pathogenesis of vascular remodeling, bone formation, and tumor progression. However, the role and underlying mechanism of CTHRC1 in post-MI wound repair are not fully clear. Bioinformatics analysis demonstrated CTHRC1 up-regulation in cardiac fibroblasts after ischemic cardiac injury. Serum levels of CTHRC1 were increased in MI mice and CTHRC1 expression was up-regulated in cardiac fibroblasts after MI. In vitro results showed that the induction of CTHRC1 expression in cardiac fibroblasts was mediated by canonical TGFβ1-Smad2/3 signaling axis. Moreover, CTHRC1 improved wound healing and boosted cardiac fibroblast activation in vitro. Cthrc1 deficiency aggravated cardiac function and reduced collagen deposition as well as increased mortality attributable to cardiac rupture after MI. Consistent with above phenotypes, reduced the levels of myocardial CD31, α-smooth muscle actin, collagen I, and collagen III was observed, whereas myocardial expression of matrix metalloproteinase 2 and matrix metalloproteinase 9 were increased in Cthrc1 knockout mice post-MI. Above effects could be partly reversed by rCTHRC1 protein or rWNT5A protein. Our study indicates that cardiac fibroblast-derived, canonical TGFβ1-Smad2/3-dependent CTHRC1 could improve wound repair and prevent cardiac rupture after MI via selectively activating non-canonical WNT5A-PCP signaling pathway.


MI model was induced by a permanent ligation of the left anterior descending (LAD)
coronary artery using the minimal thoracotomy approach described by Gao et al [4]. In brief, male mice aged 8-10 weeks old were anesthetized with 2% isoflurane gas inhalation using an isoflurane delivery system. A small skin cut was made over the left chest, and the LAD was ligated using a 6-0 silk suture at a site approximately equal to 3 mm from its origin. Sham-treated animals underwent an identical surgical procedure without LAD occlusion. Mice that died during surgery or died immediately after surgery as well as ejection fraction (EF)>50% within 24 h after MI were excluded from the present study. For survival analysis, each mouse found dead was subjected to postmortem examination and cardiac rupture was confirmed by the presence of a blood clot in the chest cavity and/or around the pericardium.

Histology
Heart tissue samples were harvested under anesthesia and perfused with PBS followed by fixation with 4% paraformaldehyde as well as embedded in paraffin and cut into 5 μm thick transverse sections at different levels. The sections were stained 3 with masson's trichrome and picrosirius red staining at apical and papillary level and then examined under ordinary polychromatic light to evaluate extent of cardiac fibrosis. The collagen volume fraction was calculated as the ratio of positive staining area to the total scar area and assessed in 10 to 15 randomly selected fields in each section. To determine the infarct size after MI, sequential cross sections of heart that were chopped above the level of the suture and 0.5 mm, 1.0 mm, 1.5 mm, as well as 2.0 mm distal to the ligature were performed with masson's trichrome staining. The infarct size was calculated as the percentage of necrotic area respect to total left ventricle area. The wall thickness of the scars at apical and papillary level was also measured. Alternatively, hearts were harvested and immediately frozen on dry ice.
The frozen hearts were cut transversely into five slices of equal thickness and stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC, T8877, Sigma-Aldrich) in PBS in a 37 °C water bath for 20 minutes. After fixation in 10% neutral buffered formaldehyde for 4 to 6 h, each slice was photographed. Infarct tissues stained appeared pale white, viable myocardium appeared brick red. The infarct size was expressed as the percentage of infarct area respect to total left ventricle area. These data were measured and analyzed using Image J software.

Immunohistochemistry
For immunohistochemistry analyses, the paraformaldehyde-fixed, paraffin-embedded sections of the heart tissue specimens were deparaffinized in xylene and rehydrated in a graded ethanol series. After antigen retrieval with citrate buffer (PH 6.0), blocking was performed with 3% BSA at room temperature for 30 min. A solution of 3% H2O2 was used to block endogenous peroxidase activity. The slices were then incubated with primary antibody against CTHRC1 (ab85739, Abcam) and vimentin (ab8978, Abcam) overnight at 4 °C. After rinsing for three times in PBS, appropriate secondary antibodies were added and incubated at room temperature for 1 h, followed by color development with DAB kit (K5007, DAKO). Images were acquired with a microscope. 4 Immunofluorescence staining were performed on the paraffin-embedded sections of the heart tissue samples fixed with 4% paraformaldehyde. After deparaffinization, antigen retrieval with citrate buffer (pH 6.0), and blocking with 3% BSA at room temperature for 30 min, the sections were incubated with these antibodies overnight at 4 °C. Afterwards, the slices were washed with PBS for three times followed by incubation with appropriate fluorescent-labeled secondary antibodies for 1 h at room temperature. Nuclei were counterstained with DAPI, which was followed by visualization using a microscope. The primary antibodies used for immunofluorescence were as follows: CTHRC1 (ab85739, Abcam), vimentin (ab8978, Abcam), cTnI (ab47003, Abcam), CD31 (ab281583, Abcam).

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from heart tissues or primary isolated cardiac cells using

Enzyme linked immune sorbent assay (ELISA)
Mouse or human blood samples were collected and centrifuged at 3000rpm for 10 minutes, followed by serum collection and storage at -80 °C. The levels of CTHRC1 were measured using commercially available ELISA Kits (CSB-EL006162MO, CUSABIO), according to the manufacturer's instructions. After adding stop solution, the absorbance was measured at 450 nm using a spectrophotometer.

Adult mouse cardiomyocytes isolation
Adult cardiomyocytes were isolated from the ventricles of male WT mice using a simplified, langendorff-free method [5]. Briefly, WT mice aged 8-10 weeks were anesthetized and the chest cavity was opened to fully expose the heart. The 6 descending aorta was chopped and the heart was immediately flushed by injecting 7 mL ethylenediamine tetraacetic acid (EDTA) buffer into the right ventricle. The ascending aorta was clipped and the heart was transferred into a 6 cm dish containing fresh EDTA buffer. Digestion was completed through sequential injection of 10 mL

Neonatal mouse primary cardiac fibroblasts isolation
Primary cardiac fibroblasts were obtained from WT neonatal mice at 1-3 days of age.
The hearts were removed from the pups immediately after euthanasia and washed with ice-cold PBS. The heart tissues were cut into about 1 mm 3 and then digested with 0.125% trypsin (25200072, Gibco) in PBS at 37 °C for 20 min/cycle (3-4 cycles).
After each cycle, the supernatant containing the isolated cells was transferred into a centrifuge tube and FBS was added at a final concentration of 10% to stop enzyme digestion. Afterwards, the supernatant was discarded after centrifuged at 1000 rpm for 5 min and the isolated cells were resuspended in DMEM with 10% FBS. All collected cell suspensions were passed through a 75 μm filter and plated on a 10 cm dish in 5% CO2 at 37 °C to allow fast-adherent cells (mainly primary cardiac fibroblasts) to attach. After 1.5 h, the medium was replaced with fresh, prewarmed DMEM. The 2nd passage primary cardiac fibroblasts were used for further experiments to eliminate other nonmyocytes.

Culture and treatment of mouse primary cardiac fibroblasts
The outgrown primary cardiac fibroblasts were cultured in DMEM containing 10% FBS, 100 U/mL penicillin, as well as 100 μg/mL streptomycin (10378016, Gibco) at 37 °C in the atmosphere containing 5% CO2. Before the initiation of the stimulation, the cells were starved in DMEM without FBS for 12 h. Afterward, they were treated with different concentrations of TGF-β1 (0 ng/ml, 2 ng/ml,5 ng/ml,10 ng/ml, and 20 ng/ml, 100-21, Pepro Tech) for 24 h. Also, they were stimulated by 10 ng/ml of TGF-β1 at different time points (0 h, 1 h, 3 h, 6 h, 12 h, and 24 h). Additionally, primary cardiac fibroblasts were treated with 10 ng/mL of TGF-β1 in the presence or absence of 10 μM of LY2109761 (HY-12075, MCE) or E-SIS3 (HY-13013, MCE) for 24 h. Due to the high homology between mouse Cthrc1 and human CTHRC1, 1 ug/ml of rCTHRC1 protein was used to treat mouse primary cardiac fibroblasts for 1 h or 24 h. All these cells were harvested for western blotting or immunofluorescence staining.
Wound healing assay 8 The migratory ability of cardiac fibroblasts was evaluated by scratch assay. Primary cardiac fibroblasts were isolated from WT mice, treated or untreated with rCTHRC1 protein. A linear wound area was created by scratching using a 200 μL pipette tip.
The plate was then gently washed to remove detached primary cardiac fibroblasts.
The migratory capacity of the cardiac fibroblasts was estimated as the covered area of the scratch repopulated by primary cardiac fibroblasts after 24 h.

Cell immunofluorescence staining
After stimulated with TGFβ1 or rCTHRC1 protein, primary cardiac fibroblasts growing on the glass side were fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS for three times, cells were permeated with 0.2% Triton X-100 in PBS for 5 min at room temperature, washed, and then blocked in 5%

Clinical study participants
A total of 40 patients who presented to our hospital with AMI were consecutively enrolled in this study. The diagnosis was made based on the following three criteria: a typical history, electrocardiographic findings in conformity to Q-wave or non-Q-wave, as well as an increase in serum myocardial enzyme levels to at least 50% above the upper normal limit. They were all treated with percutaneous coronary intervention and optimal medical treatment. Besides, 40 stable patients with confirmed normal coronary artery who underwent coronary angiography were also recruited to act as a control group. Those with significant concomitant diagnosis such as cancer, serious infections, or autoimmune diseases were excluded from the present study. The baseline clinical characteristics were summarized in Table S1. Blood samples were 9 obtained from control subjects and AMI patients at day 7 after MI. This study was approved by the ethics committee of Shanghai Jiao Tong University Affiliated Sixth People's Hospital, China [Approval no. 2021-KY-108 (K)].

Data mining and bioinformatics analysis
The GEO dataset GSE95755 used in this study to screen genes that were associated with fibrosis was available at https://www.ncbi.nlm.nih.gov/gds/. We selected fibrosis-related genes that exhibited a minimum fold-change of 2 and corrected-P value <0.05 in cardiac fibroblasts post-MI. We then identified fibrosis-related genes according to the following Gene Ontology classes: GO:0044420 (extracellular matrix part), GO:0005581 (collagen), GO:0005578 (proteinaceous extracellular matrix), and GO:0031012 (extracellular matrix).

RNA sequencing and bioinformatic analysis
To investigate possible changes in transcriptome profile elicited by Cthrc1 knockout, total RNA was extracted from WT versus C1KO left ventricle tissue 7 days after MI using the Trizol Reagent (T9424, Sigma-Aldrich), according to the manufacturer's instructions. Then the concentrations and qualities of RNA samples were determined using a NanoDrop spectrophotometer. Total RNA was used to build a complementary DNA library using the VAHTS ® Universal V6 RNA-seq Library Prep Kit for Illumina (NR604-02, Vazyme) according to the kit's instructions, after which the library was sequenced on a NovaSeq 6000 platform (Illumina). Afterwards, the Raw data was quality controlled with Skewer and FastQC, aligned with STAR, and counted with StringTie to the mouse genome (Mm10). DESeq2 was used to identify differential expression of genes. The genes that met the ± 2-fold change and corrected-P value <0.05 were further analyzed. Finally, the enrichment analyses of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed using topGO.

Statistical analysis
All values are presented as the mean± standard error of the mean (SEM) of independent experiments or independent samples with given n sizes. Statistical 1 analysis was performed with GraphPad Prism 7.0 (Graph Pad Prism Software, Inc, San Diego, CA). The normality of data distribution was determined by Shapiro-Wilk test. For comparisons, statistical analysis was performed using unpaired two tailed Student's t test (for comparing 2 groups), one-way or two-way ANOVA (for ≥3-group comparisons with one independent variable or two independent variables) with Bonferroni multiple comparison test for normally distributed data, and Mann-Whitney U test (for comparing two groups) or Kruskal-Wallis test (for multi-group comparisons) for non-normally distributed data. Survival rate was evaluated using the Kaplan-Meier method and compared by log-rank test. The difference of the total cardiac rupture frequency between two groups was assessed using χ 2 test. The values identified as outlier were excluded from statistical analysis. P <0.05 was considered statistically significant.